Resurgence of tuberculosis and other mycobacterial infections associated with AIDS threaten human health world-wide. Emergence of drug resistant TB makes it critical to discover new drug targets. Mycobacterial cell walls contain a variety of lipids with many types of multiple methyl-branched fatty acids that are unique to pathogenic mycobacteria. These lipids are thought to play important roles in the ability of the pathogens to evade the defense reactions of the host. Biosynthesis of these unique lipids containing multiple-methyl branched cell wall lipids could offer ideal targets for new antimycobacterial drugs. To elucidate the mechanisms involved in the biosynthesis of these lipids and to test for their possible functions in host-pathogen interaction, we propose the following specific aims: 1) Determine whether ORF2 expression is required for mycocerosyl transfer from mycocerosic acid synthase to phthiocerol and phenolphthiocerol in vivo by disruption of the orJ2 gene and examination of the consequence on mycocerosyl lipid biosynthesis. If ORF2 is found to be necessary, we will elucidate the mechanism of its participation in mycocerosyl lipid synthesis and determine the structure of ORF2. 2) Elucidate the nature of short-chain mycocerosic acid synthase by cloning and sequencing the gene to identify the active site domain organization and subunit composition. 3) Knock-out the gene that encodes short-chain mycocerosic acid synthase in M. tuberculosis and determine the biochemical and functional consequences. 4) Purify a newly-found branched fatty acid synthase, identify the products generated, seek the identity of the gene that encodes the synthase in the genomic data bank using the protein sequence, and determine the biochemical and functional consequences of knocking-out this gene. 5) Determine the functions of Class 1 mas-like genes, msl1 and msl2, of M. tuberculosis by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 6) Determine the functions of Class 2 (msl3, msl4, and msl5) and other mas-like genes in M. tuberculosis genome, by characterization of their products expressed in M. smegmatis, and by determination of the biochemical and functional consequences of their disruption. 7) Determine whether lack of specific lipids caused by the above gene-disruptions affects host-pathogen interaction and virulence. The results will help identify cell wall lipids critical for pathogenesis that might be suitable targets for new antimycobacterial drugs.